Antisense molecules for treatment of staphylococcus aureus infection

ABSTRACT

Disclosed are antisense molecules and compositions for the treatment of Staphylococcus aureus infection. The antisense molecules and compositions comprise nucleic acid molecules, such as RNA, DNA, or nucleic acid molecules with modified backbones, such as PNA. The antisense molecules and compositions inhibit gene expression in Staphylococcus aureus; are optionally conjugated to cell penetration molecules such as peptides; and are optionally administered in the form of a nanoparticle composition.

GOVERNMENT INTEREST

This work is based in part by the Defense Advanced Research ProjectAgency under Phase I SBIR contract number W911QX-12-C-0072. The USgovernment has certain rights to the invention.

FIELD OF THE INVENTION

The field of the invention relates to antisense polynucleotide reagentstargeting ribosomal protein expression and useful for treatment ofStaphylococcus aureus infection.

SUMMARY OF THE INVENTION

Provided are antisense molecules useful for treatment of Staphylococcusaureus infection and the inhibition of Staphylococcus aureus growth. Theantisense molecules target Staphylococcus aureus ribosomal proteins andmay comprise natural nucleic acid polymers and non-natural nucleic acidpolymers. Non-natural nucleic acid polymers include polymers withmodified backbones, such as PNA, PMO, and synthetically-modified DNA andRNA. The invention includes any type of synthetically-modified DNA orRNA that hybridizes to natural DNA and RNA. In one embodiment, theantisense molecules are in the form of a salt or a complex. In oneembodiment, the antisense molecule is complexed to a cationic polymericmolecule. In another embodiment, the antisense molecule is conjugated toa cell penetrating molecule. Also provided are pharmaceuticalcompositions comprising the antisense molecules of the invention.

In one embodiment the invention provides an antisense molecule or saltthereof that inhibits the growth of Staphylococcus aureus comprising apolynucleotide sequence that is antisense to the coding region of aStaphylococcus aureus ribosomal protein and hybridizes to said codingregion under physiological conditions. In one embodiment, the antisensemolecule is 10 to 50 nucleobases in length. In another embodiment, theantisense, molecule is fully complementary to a coding region of astaphylococcus aureus ribosomal protein. In another embodiment, theantisense molecule is at least 80% identical to a sequence selected fromthe group consisting of SEQ ID NOS: 1-50. In another embodiment, theantisense molecule is an oligonucleotide. In another embodiment, theantisense molecule is substantially pure. In another embodiment, theantisense molecule comprises a modified backbone. In another embodiment,the modified backbone is a PNA backbone. In another embodiment, theantisense molecule inhibits expression of LSU ribosomal protein L15p(L27Ae) or SSU ribosomal protein S17p (S11e). In another embodiment, theantisense molecule is conjugated to a cell penetration molecule. Inanother embodiment, the cell penetration molecule is a peptide. Inanother embodiment, the peptide is a cell-penetrating peptide (CPP). Inanother embodiment, the antisense molecule is complexed to a deliverypolymer. In another embodiment, the delivery polymer is a cationic blockcopolymer comprising phosphonium or ammonium ionic groups.

The invention also provides a method of inhibiting the growth ofStaphylococcus aureus, comprising administering an effective amount ofan antisense molecule or composition of the invention to a tissuecontaining said Staphylococcus aureus or suspected of containingStaphylococcus aureus. In one embodiment, the administering, is topicaladministration.

The invention also provides a method of treating Staphylococcus aureusinfection, comprising administering to an animal in need thereof aneffective amount of the antisense molecule or composition of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. MRSA in vitro studies. Efficacy of antibacterial nucleic acidagents is demonstrated. Peptide-PNA was tested against bacteria inculture of MRSA USA 300. Vancomycin was used to standardize theseresults for additional studies.

FIG. 2A-2B. a) MRSA fluorescent overlays 2 hours post treatment with 1uM of FITC-peptide agents. b) AcB fluorescent overlays 2 hours posttreatment with 1 uM of FITC-peptide agents. Scale bar=100 um. Thisfigure shows that the cell-penetrating peptides are non-toxic when usedalone.

FIG. 3. Shows log-phase MRSA growth inhibition over 8 hours at 0 μM, 1μM, 10 μM, and 20 μM concentration of PNA-peptide antisense antibiotic.Bottom left represents a positive control (FmhB) and bottom right showsa negative control.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The polynucleotide sequences in the sequence listing include the codingsequences for Staphylococcus aureus ribosomal proteins. See, SEQ ID NOS:65-101.

The polynucleotide sequences in the sequence listing also includeantisense deoxyribonucleic acids (DNA) and/or modified nucleic acids,such as peptide nucleic acids (PNA). These sequences are capable ofknockdown of expression of at least the following Staphylococcus aureusribosomal protein as set forth in Table 1:

TABLE 1 Antisense Polynucleotides Targeting Ribosomal ProteinsProtein Target Antisense Polynucleotide SequenceLSU ribosomal protein L10p (P0) AGACATTCAGACACC (SEQ ID NO: 21)LSU ribosomal protein L11p (L12e) TAGCCACGATGTGCA (SEQ ID NO: 19)LSU ribosomal protein L13p (L13Ae) ACGCATAATAAT (SEQ ID NO: 8)LSU ribosomal protein L13p (L13Ae) TTGACGCATAATAAT (SEQ ID NO: 33)LSU ribosomal protein L14p (L23e) GTTGGATCATTA (SEQ ID NO: 13)LSU ribosomal protein L14p (L23e) TGGATCATTAGTTAA (SEQ ID NO: 42)LSU ribosomal protein L15p (L27Ae) TTTCATTTCGGCACC (SEQ ID NO: 1)LSU ribosomal protein L16p (L10e) GGTAGTAACATTATT (SEQ ID NO: 43)LSU ribosomal Protein L18p (L5e) GATCATTTCAATACT (SEQ ID NO: 38)LSU ribosomal protein L19p TGATTTGTCATTATA (SEQ ID NO: 25)LSU ribosomal protein L1p (L10Ae) TTAGCCATTTATAGT (SEQ ID NO: 20)LSU ribosomal protein L20p ACTCGTGGCATA (SEQ ID NO: 6)LSU ribosomal protein L21p AGCAAACATACTTTG (SEQ ID NO: 31)LSU ribosomal protein L22p (L17e) TTCCATTAGGATGTC (SEQ ID NO: 45)LSU ribosomal protein L23p (L23Ae) TTCCATTATCCGAGC (SEQ ID NO: 48)LSU ribosomal protein L27p AACATCGGAATG (SEQ ID NO: 5)LSU ribosomal protein L27p TAACATCGGAATGCA (SEQ ID NO: 29)LSU ribosomal protein L28p TGTTTACCCATA (SEQ ID NO: 4)LSU ribosomal protein L2p (L8e) TAGCCATTGTCG (SEQ ID NO: 16)LSU ribosomal protein L2p (L8e) AGCCATTGTCGCTTA (SEQ ID NO: 47)LSU ribosomal protein L30p (L7e) TTTAGCCATAACTAG (SEQ ID NO: 36)LSU ribosomal protein L32p TACTGCCATGATATA (SEQ ID NO: 24)LSU ribosomal protein L34p GTTTTACCATGCAAA (SEQ ID NO: 50)LSU ribosomal protein L3p (L3e) CATCGAAAGTCC (SEQ ID NO: 17)LSU ribosomal protein L3p (L3e) GGTCATCGAAAGTCC (SEQ ID NO: 49)LSU ribosomal protein L5p (L11e) CGGTTCAAAGTGGGA (SEQ ID NO: 41)LSU ribosomal protein L6p (L9e) TCATGTTATGGC (SEQ ID NO: 12)LSU ribosomal protein L6p (L9e) ACTCATGTTATGGCA (SEQ ID NO: 39)ribosomal protein L7Ae family protein TATACTCATTTTGGG (SEQ ID NO: 26)SSU ribosomal protein S11p (S14e) TTACGTGCCATT (SEQ ID NO: 9)SSU ribosomal protein S11p (S14e) TTTACGTGCCATTTA (SEQ ID NO: 34)SSU ribosomal protein S12p (S23e) GTTGGCATGTGATAT (SEQ ID NO: 22)SSU ribosomal protein S13p (S18e) TACGTGCCATAT (SEQ ID NO: 10)SSU ribosomal protein S13p (S18e) TACGTGCCATATTAA (SEQ ID NO: 35)SSU ribosomal protein S14p (S29e) TTTAGCCACTTAATT (SEQ ID NO: 40)Zinc-dependent SSU ribosomal protein S15p (S13e)AAATTGCCATAATCA (SEQ ID NO: 27) SSU ribosomal protein S17p (S11e)TCTTTCGCTCAC (SEQ ID NO: 14) SSU ribosomal protein S17p (S11e)CGCTCACTTTTGTAA (SEQ ID NO: 2) SSU ribosomal protein S19p (S15e)TACGAGCCATTT (SEQ ID NO: 15) SSU ribosomal protein S19p (S15e)GAGCCATTTGGGCGC (SEQ ID NO: 46) SSU ribosomal protein S21pTTTAGACATCTGTAT (SEQ ID NO: 28) SSU ribosomal protein S3p (S3e)TTGACCCACAGTATT (SEQ ID NO: 44) SSU ribosomal protein S4p (S9e)CGAGCCATAATA (SEQ ID NO: 7) SSU ribosomal protein S4p (S9e)GAGCCATAATAAGAC (SEQ ID NO: 32) SSU ribosomal protein S5p (S2e)CGAGCCATGTAT (SEQ ID NO: 11) SSU ribosomal protein S5p (S2e)CGAGCCATGTATTTG (SEQ ID NO: 37) SSU ribosomal protein S6pGTTCTCATTTTATAT (SEQ ID NO: 18) SSU ribosomal protein S7p (S5e)ACGAGGCATAAT (SEQ ID NO: 3) SSU ribosomal protein S7p (S5e)TTTACGAGGCATAAT (SEQ ID NO: 23) Potential ribosomal proteinCAGTAATCATAATAA (SEQ ID NO: 30)

The sequence listing also contains control sequences of tRNA-dependentlipid II glycine ligase (FmhB): ttttccatgatttat (SEQ ID NO: 62), andNoncoding negative control (NC): aacattttggttttt (SEQ ID NO: 63).

The peptide sequences in the sequence listing include peptides thattarget and/or localize nucleic acids and nanoparticles to bacterialcells and promote bacterial membrane permeation. See Table 2:

TABLE 2 Cell Penetrating Peptides Peptide Name Amino Acid SequenceKFF peptide KFFKFFKFFK (SEQ ID NO: 51) RFF peptideRFFRFFRFFR (SEQ ID NO: 52) Magainin 2GIGKWLHSAKKFGKAFVGEIMNS (SEQ ID NO: 53) Transportin 10AGYLLGKINLKALAALAKKIL (SEQ ID NO: 54) IndolicidinILPWKWPWWPWRR (SEQ ID NO: 61) TAT peptide GRKKRRQRRRPQ (SEQ ID NO: 60)PENETRATIN 1 peptide RQIKIWFQNRRMKWKK (SEQ ID NO: 59)amphipathic peptide LLIILRRRIRKQAHAHSK (SEQ ID NO: 58)cyclic d,1-alpha-peptide KQRWLWLW (SEQ ID NO: 57)cyclic d,1-alpha-peptide RRKWLWLW (SEQ ID NO: 56)cyclic d,1-alpha-peptide KKLWLW (SEQ ID NO: 55)

Definitions

The terms used in this disclosure have ordinary meanings as used in theart.

A polymer is a linear chain of units called monomers. In a polymer, themonomeric units may be identical or they may be different. Polymers maybe natural (made in nature) or may be synthetic. Polymers of the presentinvention comprise nucleic acid polymers, polypeptides, and syntheticdelivery polymers.

A nucleic acid is a linear polymer of nucleotides. Nucleic acids made innature contain deoxyribonucleotide (DNA) bases adenine, cytosine,guanine, and thymine; or ribonucleotide (RNA) bases adenine, cytosine,guanine, and uracil. As used herein, polynucleotide and oligonucleotiderefer to a nucleic acid molecule and include genomic DNA, cDNA, RNA, ormRNA of any length. Nucleic acid, polynucleotide, oligonucleotide areterms that, may be used interchangeably.

Modified nucleic acids are non-natural polymers that hybridize tonatural DNA and RNA with sequence specificity according to Watson-Crickbase paring rules. Examples of modified nucleic acids arephosphorothioate-oligodeoxynucleotides (PS-ODNs), locked nucleic acids(LNAs), 2′-O-methyloligoribonucleotides (2′O-Mes), phosphorodiamidatemorpholino oligonucleotides (PMOs), and peptide nucleic acids (PNAs).Modified nucleic acids have modified backbones and are generally moreresistant to degradation than natural nucleic acids. The inventionincludes any type of synthetically-modified DNA or RNA that hybridizesto natural DNA and RNA. See, e.g., U.S. Pat. Nos. 5,116,195, 5,539,082,5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, 5,786,461,5,811,232, 5,837,459, 5,874,564, 5,891,625, 5,972,610, 5,986,053,6,107,470, 6,174,870, 7,098,192, 7,696,345, 8,124,745, 8,354,093,8,357,664, Wagner et al., Nucl. Acid Res. 19:5965-71 (1991); and Koshkinet al., Tetrahedron 54:3607-30 (1998).

Antisense molecules of the invention may also be composed of non-naturalpolymers that hybridize to natural nucleic acids. Atypical nucleosidebases may also be employed, such as methylated bases, phosphorylatedbases, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine,and wyosine, among others. Examples of such antisense polymerscomprising atypical bases are disclosed in U.S. Pat. Nos. 7,875,733,7,919,612, 7,939,677, 8,314,229, 8,372,969, and 8,377,898.

The term antisense polynucleotide refers to a nucleic acid molecule thatis complementary to at least a portion of a target nucleotide sequenceof interest and hybridizes to the target nucleotide sequence underphysiological conditions. Antisense molecules specifically hybridizewith one or more nucleic acids encoding a preselected target nucleicacid. The terms target nucleic acid and nucleic acid encoding the targetencompass DNA encoding the target, RNA (including pre-mRNA and mRNA)transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of an antisense compound with its target nucleic acidinterferes with the normal function of the nucleic acid. This modulationof function of a target nucleic acid by compounds which specificallyhybridize to it is generally referred to as antisense. The functions ofDNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include all vital functions such,as, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in or facilitated by the RNA. The overall effect of suchinterference with target nucleic acid function is modulation of theexpression of the target. In the context of the present invention,modulation means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the form of modulation of gene expression.

Polynucleotides are described as complementary to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, aid the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity and similarity between,two sequences can be accomplished using a mathematical algorithm (seee.g., Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). In a preferred embodiment, the percent identity between twosequences is determined based on alignments generated with the Clustal Walgorithm (Thompson, J. D. et al., 1994, Nucleic acids Res.22:4673-4680). This algorithm is incorporated into many commercialsoftware packages, in this case the alignX software program in theVector NTI suite (version 8.0). Default Clustal W parameters were usedto generate pairwise alignments from which percent identity values werecalculated (gap opening penalty of 10; gap extension penalty of 0.1).The percent identity is defined as the number of identical bases dividedby the total number of bases and multiplied by 100. If sequences in thealignment are of different lengths (due to gaps or extensions), thelength of the longest sequence will be used in the calculation,representing the value for total length.

Proteins are polymers containing one or more chains of amino acidsbonded together by peptide bonds. Proteins typically fold into a threedimensional form, facilitating a biological function.

A polypeptide is a polymer of amino acids bonded together by peptidebonds. The terms protein and polypeptide and peptide are generally usedinterchangeably, although polypeptides and peptides are generallyshorter in length than proteins.

The terms charged, uncharged, cationic and anionic refer to thepredominant state of a chemical moiety at near-neutral pH, e.g. about 6to 8. In one embodiment, the term refers to the predominant state of thechemical moiety at physiological pH, that is, about 7.4. Thus, acationic backbone linkage is predominantly positively charged at pH 7.4.

The term substantially pure means that the antisense molecule issubstantially free from other materials such as other nucleic acids,proteins, lipids, carbohydrates, and other materials with which it maybe naturally associated. In one embodiment, substantially pure antisensemolecules are 95-95% homogeneous by HPLC. In another embodiment,substantially pure antisense molecules are 99-100% homogenous by HPLC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood by reference to the followingdetailed description of the embodiments of the invention and examplesincluded herein. The terminology used herein is for the purpose ofdescribing embodiments of the invention and is not intended to belimiting.

Specific aspects of the invention include antisense molecules that areuseful for the treatment of Staphylococcus aureus infection and/orinhibit the growth of Staphylococcus aureus comprising, an antisensemolecule that is antisense to a Staphylococcus aureus ribosomal proteincoding region under physiological conditions. In one embodiment, theantisense molecule hybridizes to a Staphylococcal aureus ribosomalcoding region selected from the group consisting of SEQ ID NOS: 65-101.In one embodiment the antisense molecule contains 10-50 nucleobases,i.e., is a 10-50-mer. In another embodiment, the antisense molecule is a10-25-mer, a 12-20-mer, a 12-15-mer, a 11-mer, a 12-mer, a 13-mer, a14-mer, a 15-mer, a 16-mer, a 17-mer, an 18-mer, a 19-mer, a 20-mer, a21-mer, a 22-mer, a 23-mer, a 24-mer, a 25-mer, a 26-mer, a 27-mer, a28-mer, a 29-mer, or a 30-mer. The nucleotide sequence for the antisensemolecule is chosen at a binding location that preferably spans the startcodon. Proprietary software scans window sizes 10 bases, 11 bases, 12bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19bases, and/or 20-40 bases (as a non-limiting example) including thestart codon and ranks self-folding potential by base content. The software algorithm may be programmed to span the start codon. Alternatively,the algorithm may be programmed to optionally span the start codonregion. Selection of antisense sequence can be finalized manually fromthese data or through an automated process derived from empirical dataand parameter weighting. These antisense molecules againstribosomally-expressed genes are substantially orthogonal to the humantranscriptome. In one embodiment, the antisense molecules have baselengths exhibiting features such as Tm greater than 37° C., lowself-folding, and significant start codon overlap.

In another embodiment, the invention provides a polynucleotide sequenceat least 80% identical to a sequence selected from SEQ ID NO: 1-50.Specifically, the sequences may contain one or more substitutions,additions, deletions, and/or insertions with natural or non-naturalnucleotides, such that the target gene modulation activity is notsubstantially diminished. Variants exhibit at least about 80%, 81%, 82%,83%, 84% 85%, 86%, 87%, 88%, or 89% sequence identity; and anotherembodiment at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identity to a sequence selected from the group consisting of SEQID NOS: 1-50. The percent identity may be readily determined bycomparing sequences of the polynucleotides to the corresponding portionof the target polynucleotide, using any method including using computeralgorithms well known to those of ordinary skill in the art. Algorithmsinclude the Align or the BLAST algorithm (Altschul, 1991 J. Mol. Biol.219:555-565; Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA89:10915-10919).

In one embodiment of the invention, the active ingredient is coupled toa targeting/cell penetration molecule. In one aspect of the invention,the targeting molecule comprises a peptide. The peptide may comprise acell penetration peptide (CPP). Peptides utilized may have one or morefunctions to facilitate cell targeting and/or membrane permeation. Inparticular, the therapeutic polynucleotides of the invention can bedelivered to Staphylococcus aureus in a host by conjugating peptides tothe antisense molecule. The ability to conjugate antisense molecules topeptides for membrane disruption of bacteria provides specificity andreduces toxicity. Examples of cell penetration peptides include thosehaving SEQ ID NOS: 51-61. Additional examples cell penetration peptidesand methods to link them to antisense molecules are described in U.S.Pat. Nos. 8,354,387, 8,354,093, 8,313,778, 8,299,236, 8,242,081,8,211,468, 8,207,293, 8,138,383, 8,044,019, 8,039,587, 7,943,581, and7,879,813. In another embodiment, the cell penetrating peptides isderived from HIV tat, herpes virus VP22, the Drosphila Antennapediahomeobox gene product, signal sequences, fusion sequences or protegrin Ias disclosed in U.S. Pat. No. 8,338,366. The antisense molecule-peptideconjugate may be prepared by methods of solid-phase synthesis, wherecysteine serves as the linker between peptide and DNA. Othermethodologies known in the art may be used (See for example, Dirksen,A., et al., J. Am. Chem. Soc. 2006. 128, 15602-3).

CPPs useful in the invention are peptides of diverse origins. Cationicnucleic acid carrier peptides form productive nanoparticles when mixedwith the synthetic polymers of the invention. One example is the peptideKFFKFFKFFK (SEQ ID NO: 51) described in Xie et al., Molecular Therapy2004, 10, 652-659. Additional peptides may include TAT peptide andPENETRATIN. The TAT peptide, GRKKRRQRRRPQ (SEQ ID NO: 60), is derivedfrom the transactivator of transcription (TAT) of human immunodeficiencyvirus and is a CPP. CPPs overcome the lipophilic barrier of cellmembranes and deliver large molecules and particles inside the cell fortheir biological actions. PENETRATIN peptide is a 16-amino acid peptideof sequence RQIKIWFQNRRMKWKK (SEQ ID NO 59) corresponding to the thirdhelix of the homeodomain of Antennapedia protein.

Useful CPPs also encompass cyclic d,l-αpeptides, such as, KQRWLWLW (SEQID NO: 57), RRKWLWLW (SEQ ID NO: 56), and KKLWLW, (SEQ ID NO: 55) asdescribed in Fernandez-Lopez et al., Nature 2001, 412, 452-455. Thesepeptides have antibiotic properties of their own, and also function ascarriers of cargo for internal cellular delivery. Additionally,amphipathic peptides LLIILRRRIRKQAHAHSK (SEQ ID NO: 58) and transportin10 (TP10), AGYLLGKINLKALAALAKKIL (SEQ ID NO: 54), described inNekhotiaeva et al. FASEB J. 2010, 394-396, form productivenanoparticles. Tryptophan rich peptides, such as Magainin 2 peptide,GIGKWLHSAKKFGKAFVGEIMNS (SEQ ID NO: 53), which was isolated from theAfrican clawed frog (Karas et al, Biochemistry 2002, 41, 10723-31), areadditional CPPs useful in the present invention. Furthermore,Indolicidin, ILPWKWPWWPWRR (SEQ ID NO: 61), which was isolated frombovine neutrophils, is another CPP useful in the present invention.These and other peptides of similar sequence and properties arerecognized h one of skill in the art as functional alternatives and areencompassed by the present invention. Furthermore, these peptides may bemodified to improve function as desired or needed.

Bulk peptide and polynucleotide synthesis can be carried out by contractmanufacturers, such as Neo Group, Inc. (Cambridge, Mass.) using standardmethodologies including solid-scaffold protection/deprotection synthesisvia high fidelity synthesizers. The peptide-PNA or peptide-DNA componentis the therapeutic molecule which enters the pathogen and disrupts itsgenetic regulation.

In one embodiment, an antisense molecule is conjugated to a CPP usingwell known conjugation methods that employsuccinimidyl-6-hydrazinonicotinateacetonehydrazone tosuccinimidyl-4-formylbenzoate coupling chemistry. This is a specific,well-behaved, and highly efficient conjugation method for peptide-DNAcoupling. In order to covalently couple peptides to nucleic acids, thepeptides are prepared for reaction by, modifying the N-terminal with areactive group. In one embodiment, the N-terminal of the peptide ismodified with S6H (succinimidyl-6-hydrazinonicotinateacetonehydrazone).N-protected peptides are desalted and dissolved in dry DMF. Next, S6H isadded in 2× molar excesses to a stirring, solution and allowed to reactat room temperature for 2 hours. Workup follows procedures known in theart, such as that described by Dirksen et al. J. Am. Chem. Soc. 2006128, 15602-3. Other methods of coupling peptides to nucleic acids knownin the art may be used.

An FITC assay may be utilized to monitor cellular uptake of peptides.Peptides were conjugated to fluorescein isothiocyanate (FITC) to monitoruptake using florescence microscopy. FIG. 2A-2B show assay results forseveral peptides as tested in MRSA (FIG. 2A) and AcB (FIG. 2B)(fluorescent overlays 2 hours post-treatment with 1 μM of FITC peptideagents, scale bar=100 μm). For MRSA, the helical cationic peptides withKFF and RFF motifs are effective for cellular entry. Also, Magainin-FITCis effective for entry into MRSA. There do not appear to be anybactericidal effects from the peptides at the tested concentration (1μM) in any of the micrographs presented in FIG. 2A-2B.

In another embodiment of the invention, the antisense molecule iscombined with a delivery polymer. The polymer-based nanoparticle drugdelivery platform is adaptable to a diverse set of polynucleotidetherapeutic modalities. In one aspect of the invention, the deliverypolymer is cationic. In another aspect of the invention, the deliverypolymer compiles phosphonium ions and/or ammonium ions. In anotherexample of the invention, the antisense molecule is combined with adelivery polymer, and the composition forms nanoparticles in solution.In, a further embodiment, nanoparticle polyplexes are stable in serumand have a size in the range of about 30 nm-5000 nm in diameter. In oneembodiment, the particles are less than about 300 nm in diameter. Forexample, the nanoparticles are less than about 150 nm in diameter.

In one embodiment, the delivery vehicle comprises a cationic blockcopolymer comprising phosphonium or ammonium ionic groups as describedin PCT/US12/42974. In one embodiment, the polymer isdiblock-Poly[(ethylene glycol)₉ methyl ethylmethacralate][stirylphosphoniurn]. In another embodiment of theinvention, the delivery polymer comprises glycoamidoamines as describedin Tranter et al. Amer Soc Gene Cell Ther, December 2011;polyhydroxylamidoamines, dendritic macromolecules,carbohydrate-containing polyesters, as described in US20090105115; andUS20090124534. In other embodiments of the invention, the nucleic aciddelivery vehicle comprises a cationic polypeptide or cationic lipid. Anexample of a cationic polypeptide is polylysine. See U.S. Pat. No.5,521,291.

In one embodiment, the antisense molecules are part of a compositioncomprising delivery or carrier polymers. In another embodiment, theantisense molecules are part of nanoparticle polyplexes capable oftransporting antisense molecules with stability in serum. The polyplexcompositions comprise a synthetic delivery polymer (carrier polymer) andbiologically active compound associated with one another in the form ofparticles having an average diameter of less than about 500 nm, such asabout 300 nm, or about 200 nm, preferably less than about 150 nm, suchas less than about 100 nm. The invention encompasses particles in therange of about 40 nm-500 nm in diameter.

In one embodiment, the delivery or carrier polymer comprises a cationicblock copolymer containing phosphonium or ammonium ionic groups asdescribed in PCT/US12/42974. In another embodiment of the invention, thedelivery or carrier polymer comprises glycoamidoamines as described inTranter et al. Amer Soc Gene Cell Ther, December 2011:polyhydroxylamidoamines, dendritic macromolecules,carbohydrate-containing polyesters, as described in US20090105115; andUS20090124534. The polyglycoamidoamine (PGAA) polymer system, which is aproprietary, localized and biodegradable nanoparticle system, representsanother delivery or carrier polymer. Poly(galactaramidoamine) is anefficient cationic polymeric vehicle with low cytotoxicity(Wongrakpanich et al. Pharmaceutical Development and Technology, Jan.12, 2012). The nanoparticle delivery system disclosed in Hemp et al.Biomacromolecules, 2012 13:2439-45 represents another delivery orcarrier polymer useful in the present invention.

In other embodiments of the invention, the delivery or carrier polymercomprises a cationic polypeptide or cationic lipid. Polymers, such aspoly-L-lysine (PLL), polyethyleneimine (PEI), chitosan, and theirderivatives are also encompassed by the invention. Nucleic acid deliveryusing these compounds relies on complexation driven by electrostaticinteractions between the gene and the polycationic delivery agent.Polymer DNA complexes condense into particles on the order of 60 nm-120nm in diameter. Polymers such as, linear PEI and PLL have hightransfection rates in a variety of cells.

In vivo nucleic acid delivery has size constraints requiring asufficiently small polyplex to enable long circulation times andcellular uptake. In addition, polyplexes must resist salt- andserum-induced aggregation. Serum stability is generally associated witha particle size of about sub-150 nm hydrodynamic radius or belowmaintainable for 24 h. The nanoparticles of the invention, whichcomprise nucleic acid therapeutic and delivery polymer, have thehydrodynamic radius and material properties for serum stability. Inparticular, the delivery polymer, when combined with the nucleic acid,protects the therapeutic cargo under physiological conditions. Thedelivery polymers are designed to have characteristics of spontaneousself-assembly into nanoparticles when combined with polynucleotides insolution.

The invention also contemplates other delivery polymers that formserum-stable nanoparticles. The invention is not limited to the type ofdelivery polymer and may be adaptable to nucleic acid characteristics,such as length, composition, charge, and presence of coupled peptide.The delivery polymer may also be adaptable for material properties ofthe resultant nanoparticle, such as hydrodynamic radius, stability inthe host bloodstream, toxicity to the host, and ability to release cargoinside a host cell.

In one embodiment, the antisense molecule or penetrating peptideconjugate thereof is administered in the form of a salt. The salt may beany pharmaceutically acceptable salt comprising an acid or base additionsalt. Examples of pharmaceutically acceptable salts with acids includethose formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid,hydrofluoric acid, phosphorous acid, and the like. Also included aresalts that are formed with organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, alkanedioic acids, aromatic acids, aliphatic and, aromaticsulfonic acids, etc. and include, for example, acetic acid,trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Exemplary salts thus include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates,trifluoroacetates, propionates, caprylates, isobutyrates, oxalates,malonates, succinate suberates, sebacates, fumarates, maleates,mandelates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates,phenylacetates, citrates, lactates, malates, tartrates,methanesulfonates, and the like. Also contemplated are salts of aminoacids, such as arginates, gluconates, and galacturonates (see, forexample, Berge S. M. et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Science, 66:1-19 (1997). Acid addition salts of basicantisense molecules may be prepared by contacting the free base formswith a sufficient amount of the desired acid to produce the saltaccording to methods and techniques with which a skilled artisan isfamiliar.

Pharmaceutically acceptable base addition salts are formed by additionof an inorganic base or an organic base to the free acid.Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Salts derived from inorganic bases include, but are not limitedto, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum salts and the like. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, for example, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, diethanolamine,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine,N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline,betaine, ethylenediamine, ethylenedianiline, N-methylglucamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.

The antisense molecules are administered as part of a pharmaceuticalcomposition comprising a pharmaceutically acceptable diluent, excipientor carrier. Suitable diluents, excipients and carriers are well known inthe art and are described, for example, in Remington's PharmaceuticalSciences, Mack Publishing Co. (A. R. Gernnaro Ed., 1985). Thepharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,saline, ethanol, a polyol (for example, glycerol, propylene, glycol,liquid polyethylene glycols, and the like), vegetable oils, nontoxicglyceryl esters, and suitable mixtures thereof. The proper fluidity canbe maintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the antisensemolecule in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

The invention also provides a method of treating Staphylococcus aureusinfection and a method of inhibiting the growth of Staphylococcusaureus. In one embodiment, the Staphylococcus aureus is amethicillin-resistant (MRSA) strain. In one embodiment, the animalundergoing treatment for Staphylococcus aureus infection exhibits one ormore symptoms of Staphylococcus aureus infection including pussproduction in the infected area, boils, abscesses, carbuncles, stys,and/or cellulitis. The animal may also exhibit signs of sepsis orpneumonia.

In one embodiment, the antisense molecules are administered byintravenous, intramuscular, or peritoneal injection. In anotherembodiment, the antisense molecules are administered topically, e.g. toa tissue suspected to be infected by Staphylococcus aureus. In anotherembodiment, the antisense molecules are administered orally. Whenadministered orally, the antisense molecules may be formulated as partof a pharmaceutical composition coated with an enteric coating that willprotect the antisense molecules from the acid environment of the stomachand release the antisense molecules in the upper gastrointestinal tract.In another embodiment, the antisense molecules may be formulated as,part of a sustained release formulation that will release the antisensemolecules on a substantially continuous basis over a period of time.

Animals that may be treated with the antisense molecules according tothe invention include any animal that may benefit from treatment withthe antisense molecules. Such animals include mammals such as humans,dogs, cats, cattle, horses, pigs, sheep, goats and the like.

The antisense molecules are administered in an amount that is effectivefor the treatment of Staphylococcus aureus infection or inhibition ofthe growth of Staphylococcus aureus. The amount may vary widelydepending on the mode of administration, the age of the animal, theweight of the animal, and the surface area of the mammal. The amount ofantisense molecule, conjugate, salt and/or complex thereof may rangeanywhere from 1 pmol/kg to 1 mmol/kg. In, another embodiment, the amountmay range from 1 nmol/kg to 10 mmol/kg. When administered topically, theamount of antisense molecule, conjugate, salt and/or complex, thereofmay range anywhere from 1 to 99 weight percent. In another embodiment,the amount of antisense molecule, conjugate, salt and/or complex thereofmay range anywhere from 1 to 10 weight percent.

Example I

Synthesis of Peptide-PNA Conjugate:

All PNA agents were prepared using heterogenous solid-phase peptidesynthesis techniques and purified with HPLC.

Although direct dosing with naked polynucleotides has been used toinhibit pathogenesis of MRSA in culture, a significant barrier fornucleic acid therapy in humans is the bacterial cell wall. To overcomethe cell wall barrier, peptides derived from bacterial-infectingorganisms that can penetrate these bacterial cell walls can be attachedto nucleic acids or modified nucleic acids to enhance nucleic acid entryinto the bacterium.

DNA sequences were synthesized using high-fidelity synthesizers made byNEO-Bio Group, Cambridge, Mass. The polynucleotide was then coupled topeptides which permit permeation of bacterial membranes andpolynucleotide entry. In the present invention, solid-phase syntheticmethodology for peptide-DNA coupling was employed where cysteine servedas the linker between peptide and DNA.

In a specific embodiment, antisense 15-mer DNA and PNA analogs weresynthesized for testing in cell culture. A positive control fromliterature (FmhB); and a noncoding sequence for use as a negativecontrol (NC) were also synthesized. Each polynucleotide was coupled tothe cell penetrating peptide (CPP) motif KFFKFFKFFK (SEQ ID NO: 51).

Both PNA-CPP and DNA-CPP candidates were synthesized and tested. Massspectrometric analysis of each conjugate was performed to confirmsuccessful synthesis. The purity of the PNA-peptide and DNA-peptidecandidates was established using HPLC. Purity of about 99.9% wasachieved for PNA-peptide; while >87% was achieved for DNA-peptide.DNA-peptides yielded a higher degree of impurity likely due to the stepsrequired to make the DNA and CPP peptide separately and then conjugatethem before a final purification step. Conversely, synthesis of the PNAagents yielded purity levels of about 99%. Increased purity andsimplicity of manufacture of PNA-peptide therapeutics providesadvantages over DNA-peptide candidates with respect to cGMP-compliantmanufacture in battlefield arenas.

Example II

An FITC assay was utilized to monitor cellular uptake of peptides.Peptides were conjugated to fluorescein isothiocyanate (FITC) to monitoruptake using florescence microscopy. FIG. 2A-2B shows assay results forseveral peptides as tested in MRSA (FIG. 2A) and AcB (FIG. 2B)(fluorescent overlays 2 hours post-treatment with 1 μM of FITC-peptideagents, scale bar=100 μm). For MRSA, the helical cationic peptides withKFF and RFF motifs are effective for cellular entry. Also, Magainin-FITCis effective for entry into MRSA. There do not appear to be anybactericidal effects from the peptides at the tested concentration (1μM) in any of the micrographs presented in FIG. 2A-2B.

Example III

MRSA In Vitro Studies:

Demonstration of sequence-specific effects of PNA-peptide molecules onMRSA was carried out in MRSA USA 300. MRSA USA 300 is a major source ofcommunity-acquired infections in, the US, Canada and Europe. CloneFPR3757 is a multidrug resistant USA 300 strain that is available fromATCC as both the culture (ATCC® BAA-1556TM) and the genomic DNA (ATCC®BAA-1556D-5). MRSA USA 300 strain is well characterized which allows forreliable benchmarking. MRSA growth curves were generated by inoculatingfreshly thawed frozen bacterial stocks at different dilutions rangingfrom 1:3000, 1:1500, 1:600 and 1:300 in Tryptic Soy Broth (TSB,Becton-Dickinson). Absorbance readings were taken hourly at 600 nm(A₆₀₀) and 550 nm (A₅₅₀) using, a Biomate 3S spectrophotometer (ThermoScientific) to establish optimal measurement settings and characterizebacterial growth kinetics. Readings at 550 nm gave slightly highersensitivity. There was a correlation seen with the lower dilutiontitrations and a faster time to higher absorbance value. A550 wasestablished as the optimal measurement to assess propagation invancomycin titration and Minimum Inhibitory Concentration (MIC) assays.

Vancomycin titrations were established to determine a suitable testrange. An 800 ug/ml stock solution was diluted tenfold in TSB to 80ug/ml and further serial diluted to 40, 20, 10, 5, and 2.5 μg/ml in TSB,respectively. MRSA USA 300 strain was cultured to an early log phase OD550 value of 0.111 and treated with the 80-2.5 μg/ml range ofvancomycin. Absorbance measurements at 550 nm were taken hourly over a4-hour time period.

Minimum inhibitory concentration (MIC) analyses were performed asdescribed in Clinical and Laboratory Standards Institute. Methods forDilution Antimicrobial Susceptibility Tests for Bacteria that GrowAerobically, 7th ed.; Approved Standard M7-A7; CLSI: Wayne, Pa., USA,2006; volume 26, No. 2. Vancomycin and methicillin were used ascontrols. MIC was determined as the lowest concentration of agent thatinhibits bacterial growth detected at A₆₀₀.

Time-kill analyses were performed as described in Haste et al. J.Antibiot. 2010, 63, 219-224. Agents at various concentrations werealiquoted into the Falcon tubes. Four ml of bacteria at 5E5 cfu/ml wereadded to the tubes. Tubes were incubated in a shaker at 37° C., and at0, 2, 4, and 8 h were subsequently analysed for bacterial growth viaA₆₀₀.

Sequence-specific effects of polynucleotide-peptide agents against MRSA:A wide range of concentrations were tested for the PNA-peptide antisensesequences determined from bioinformatics. FmhB was used as a positivecontrol from the literature (Xie et al., Molecular Therapy, 2004, 10,652-659) and a non-encoding sequence with a terminal (KFF)₃K motif wasused as a negative control (NC) to indicate bactericidal effectsimparted by peptide membrane disruption. Sequence-specific inhibitionwas demonstrated by treating bacteria during lag phase to determinegrowth inhibition and potential recovery at later time points. Thecandidate agents and non-coding sequence control were diluted in a rangefrom 20 μM, 5 μM, 1 μM, 250 μM, and 25 μM with sterile RNase-free,DNase-free water. Inhibition of MRSA growth was observed over a widerange of PNA-peptide concentrations.

The time course shown in FIG. 3 was carried out using MRSA strain USA300. Freshly-thawed MRSA at a 1:100 dilution in TSB was added to wellscontaining the individual PNA-peptide molecules. An additional positivecontrol, vancomycin at 12.5 ug/ml, and a negative control, water only,were also assayed. The samples were allowed to incubate at 37° C. with225 RPM orbital shaking and measured at two-hour time intervals, over an8-hour time course. As FIG. 1 illustrates, inhibition of MRSA growth wasobserved over time at a 5 μM concentration.

In log-phase growth, inhibition is observed at concentrations as low as˜1 μM for PNA-peptide conjugates (FIG. 3) and as low, as ˜10 μM forDNA-peptide conjugates.

FIGS. 2A-2B demonstrate the non-toxicity of thecell-penetrating-peptides. When conjugated to FITC and added to cells inculture, the cells remain alive over time periods of the cell cultureexperiments.

Example IV

To dissolve the DNA-peptide conjugates, they were dispersed in trisbuffer at an elevated pH=9. The conjugates were then gently agitated for24 h at 40° C. After this time period cloudiness was still observed, sothe conjugates were heated to 80° C. under gentle agitation for anadditional 6h, after which clear solutions were obtained. The initialsolution was tested via DLS to look at for potential self-assemblybetween the DNA-peptide conjugates. As exhibited with many chargedpolymers there was self-aggregation observed in solution, showing broadpolydisperse aggregates in the 300 nm to 1-micron range.

Particle size plays an important role in determining blood circulationtime and clearance. It is also a predictor of tissue permeation,clearance potential, and selectivity. Polymer-containing particles havebeen validated with siRNA and DNA, are capable of protecting nucleicacids from nuclease degradation, and can be engineered for colloidalstability in the bloodstream. The antisense molecule-peptide conjugatesof the present invention were combined with serum-stablephosphonium-block copolymers to form polyplexes. This diblock copolymerforms a supramolecular assembly with negatively-charged DNA. Theparticle forms a core-shell type morphology with a neutral polyethyleneglycol (PEG) brush on the surface. Polyplex hydrodynamic diameter wasmeasured on a Zetasizer (Nano ZS) dynamic light scattering (DLS)instrument (Malvern Instruments, Worcestershire, UK). As a sizecomparison, a DNA-peptide conjugate (S11e-KFFKFFKFFK (SEQ ID NO: 51)without carrier polymer, was measured at 1 mg/ml in tris buffer solutionat pH=9. This DNA-peptide conjugate with diblock-Poly[(ethylene glycol)₉methyl ethyl methacralate][stirylphosphonium] at three concentrationsexhibited size ranges from 40 nm-300 nm.

Formation of nanoparticles with the DNA-peptide conjugates is dependenton physical factors. Because the DNA region is negatively charged andthe KFFKFFKFFK (SEQ ID NO: 51) region is positively charged, theconjugates exhibit strong intramolecular associations in solution. Awide range of formulation conditions were evaluated. Optimal particlesform at charge-to-charge ratios of 2-4 (phosphonium+/DNA phosphate-) and[DNA-peptide conjugate]≤0.5 mg/ml and lower. When concentrations exceed0.5 mg/nil, dynamic light scattering (DLS) analysis indicates that largeaggregates form. The DLS data indicates that pre-formulationconcentration influences the final nanoparticle size range, with 0.5mg/ml forming the largest nanoparticles clustering around 90 nm-100 nm;and 0.1 mg/ml forming particles as small as 40 nm diameter.

To dissolve the DNA-peptide conjugates, they were dispersed in trisbuffer at an elevated pH=9. The conjugates were then gently agitated for24 h at 40° C. After this time period cloudiness was still observed, sothe conjugates were heated to 80° C. under gentle agitation for anadditional 6 h, after which clear solutions were obtained. The initialsolution was tested via DLS to look at for potential self-assemblybetween the DNA-peptide conjugates. As exhibited with many chargedpolymers there was self-aggregation observed in solution, showing broadpolydisperse aggregates in the 300 nm to 1 μm range.

All patents, patent applications and publications cited herein are fullyincorporated by reference.

1. An antisense molecule or salt thereof that inhibits the growth ofStaphylococcus aureus comprising a polynucleotide sequence that isantisense to the coding region of a Staphylococcus aureus ribosomalprotein and hybridizes to said coding region under physiologicalconditions, wherein said antisense molecule is 10 to 50 nucleobases inlength and has a sequence that is at least 95% identical to a sequenceselected from the group consisting of SEQ ID NOs: 3-17, 28, and 47, andwherein said antisense molecule is conjugated to a cell penetrationmolecule. 2-3. (canceled)
 4. The antisense molecule of claim 1, that hasa sequence selected from the group consisting of SEQ ID NOs: 3-17, 28,and
 47. 5. The a antisense molecule of claim 1, which is anoligonucleotide.
 6. The antisense molecule of claim 1, which issubstantially pure.
 7. The antisense molecule of claim 1, wherein theantisense molecule comprises a modified backbone.
 8. The antisensemolecule of claim 7, wherein the modified backbone is a PNA backbone.9-10. (canceled)
 11. The antisense molecule of claim 1, wherein saidcell penetration molecule is a peptide.
 12. (canceled)
 13. A compositioncomprising an antisense molecule of claim 1, complexed to a deliverypolymer.
 14. The composition of claim 13, wherein said delivery polymeris a cationic block copolymer comprising phosphonium or ammonium ionicgroups.
 15. A method of inhibiting the growth of Staphylococcus aureus,comprising administering an effective amount of an antisense molecule ofclaim 1 to a tissue containing said Staphylococcus aureus or suspectedof containing Staphylococcus aureus.
 16. The method of claim 15,comprising topical administration of the antisense molecule.
 17. Amethod of treating Staphylococcus aureus infection, comprisingadministering to an animal in need thereof an effective amount of theantisense molecule of claim
 1. 18. The method of claim 5, wherein saidantisense molecule is complexed to a delivery polymer.
 19. The method ofclaim 17, wherein said antisense molecule is complexed to a deliverypolymer.
 20. A pharmaceutical composition comprising the antisensemolecule of claim 1 and a pharmaceutically acceptable diluent,excipient, or carrier.
 21. The composition of claim 20, wherein theantisense molecule is 99-100% homogeneous by high-performance liquidchromatography (HPLC).